Although the vast majority of antibacterial are derived from microbes and new drugs are critically needed, many pharmaceutical companies have abandoned natural products because previous discovery paradigms seem no longer fruitful. However, because less than 2% of environmental microorganisms are cultivable by classical methods, the rest have not been screened. One method for accessing this potentially rich drug source is to shotgun clone large DNA fragments from uncultivated microbes, some of which may encode complete biosynthetic pathways, and screen them by a variety of biological and informatics-based methods, such as expressing the DNA in surrogate hosts and then screening for antibacterial activity. As different hosts provide different expression capabilities, using a variety of hosts is advantageous. We previously developed methods to create environmental DNA libraries by cloning large DNA fragments directly from soil samples into BAG vectors, then transferring them into 3 hosts: E. coli, S. lividans, and P. putida for expression and screening. We discovered a small environmental gene, rep, that significantly enhances type II PKS expression in streptomyces, thus increasing the chance of detecting low-level expression of environmental DNA. This proposal aims to further determine the scope of the ability of rep to enhance gene expression. Specifically, we will measure the expression of erythromycin (a type I PKS) and the calcium-dependent antibiotic (CDA, a NRPS) in the presence and absence of rep. To further enhance biodiversity and expression possibilities, we will also test the ability of rep to enhance expression of endogenous antibiotic clusters encoded by two marine microbes (S. arenicola and S. maritimus), and then assay the ability of rep to enhance expression of a marine cluster (enterocin) in S. lividans. Once the scope of expression enhancement effected by rep is determined, it can be inserted into selective expression hosts, possibly including a marine host, to optimize the detection of antibiotic biosynthetic genes in soil and marine environmental libraries. [unreadable] [unreadable]